Brazilian Journal of Medical and Biological Research (2015) 48(2): 146-153, http://dx.doi.org/10.1590/1414-431X20144129 ISSN 1414-431X

Measurement of serum and estrogen metabolites in pre- and postmenopausal women with osteoarthritis using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

W.L. Gao1*, L.S. Wu1*, J.H. Zi1,B.Wu1, Y.Z. Li2, Y.C. Song3 and D.Z. Cai3

1Department of Orthopedics, Linyi People’s Hospital of Shandong Province, Linyi, China 2Guangdong Institute for Drug Control, Guangzhou, Guangdong, China 3Department of Orthopedics, the Third Affiliated Hospital of Southern Medical University, Guangzhou, China

Abstract

Although 17b- (E2) deficiency has been linked to the development of osteoarthritis (OA) in middle-aged women, there are few studies relating other and estrogen metabolites (EMs) to this condition. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method to measure the levels of six EMs (i.e., , E2, , 2-hydroxyestrone, 2-hydroxyestradiol, and 16a-hydroxyestrone) in healthy pre- and postmenopausal women and women with OA. This method had a precision ranging from 1.1 to 3.1% and a detection limit ranging from 10 to 15 pg. Compared to healthy women, serum-free E2 was lower in the luteal and postmenopausal phases in women with OA, and total serum E2 was lower in postmenopausal women with OA. Moreover, compared to healthy women, total serum 2-hydroxyestradiol was higher in postmenopausal women with OA and total serum 2-hydroxyestrone was lower in both the luteal and follicular phases in women with OA. In conclusion, our HPLC-ESI-MS/MS method allowed the measurement of multiple biochemical targets in a single assay, and, given its increased cost-effectiveness, simplicity, and speed relative to previous methods, this method is suitable for clinical studies.

Key words: Estrogen; Estrogen metabolites; Menopause; Serum; Osteoathritis

Introduction

Estrogens are endogenous female hormones required , respectively, in a reaction catalyzed by for the growth and development of target tissues, including aromatase, which is expressed primarily the mammary gland, and which play a major role in the in ovarian granulosa cells, adipose stromal cells, and the etiology of breast cancer (1,2). Estrogen has placenta. Circulating E2, the primary estrogen in premen- been related to osteoporosis and increased fracture risk opausal and early perimenopausal women, declines with (3,4), and lower baseline serum 17b-estradiol (E2)and menopause. E1 generated by oxidation of E2 is hydrox- urinary 2-hydroxyestrone (2-OHE1) levels have been linked ylated via two mutually exclusive pathways (6) to generate to knee osteoarthritis (OA) in middle-aged women (5). That either 2-hydroxyestrone (2-OHE1) or 16a-hydroxyestrone said, there are relatively few studies linking serum levels of (16a-OHE1; Figure 1), which are estrogen agonists (7) with other estrogens and estrogen metabolites (EMs) to the target tissue-specific biological activities distinct from one pathogenesis of female OA. another and from E2. Compared to E2, 16a-OHE1 binds Endogenous estrones (E1) and E2 are synthesized with lower affinity to both the (8) and to from the androgenic precursors and serum -binding globulin, and is consequently

Correspondence: Wanli Gao: ,[email protected]..

*These authors contributed equally to this study.

Received May 15, 2014. Accepted September 9, 2014. First published online November 14, 2014.

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Chromatographic separation removes most of the interfer- ences caused by impurities, and multiple co-eluting moieties with distinct molecular masses and chemical structures generate characteristic fragments and specific mass spec- tra. Although LC/MS methods (19-21) measure more biochemical targets in a single assay than HPLC-ESI-MS/ MS, the multiple reaction-monitoring quantitative model of HPLC-ESI-MS/MS allows the identification and quantifica- tion of products in the picogram range in only 10 mL of blood. We established an HPLC-ESI-MS/MS method to determine serum levels of 6 EMs in pre- and postmeno- pausal women with OA and in healthy women (Figure 2).

Figure 1. Estrogen metabolism pathways, including estrone, Material and Methods estradiol, and estrogen metabolites. 17b-HSD: 17beta-hydroxy- dehydrogenase; P450 AROM: cytochrome P450 aromatase. Equipment The following equipment was used: TI-H-15 supersonic more available in estrogen-sensitive target tissues than E2. generator (ELMA, Germany), incubator (LRH-250A, Although 2-hydroxyestrogens have a reduced binding Guangdong Province Medical Instrument Factory, China), affinity for the estrogen receptor compared to 16a-OHE1, LABORTA 4003 rotary evaporation (Heidolphon, Germany), they participate in oxidation/reduction reactions (9,10). U3 aqueous bath (Julabo, Germany), LRH-250A homeo- A variety of analytical methods have been developed thermia incubator (Medical Apparatus and Instruments to analyze EMs, including spectrophotometry (11) and Factory, China), Mili-Q deionized water deviser (Millipore, thin-layer chromatography (12) for high EM levels, and USA), ME-5 electronic balance (Sartorius, Germany), radioimmunoassay (RIA) (13,14) and -linked immu- API3000 tandem quadrupole axle mass spectrometer (AB nosorbent assay (15) for trace EM levels. Given the Company, USA), and an Agilent 1100 liquid chromatography problems of cross-contamination associated with these system (Agilent, USA), ZORBAX C18 chromatographic techniques, however, gas chromatography (GC) (16) and column (4.66250 mm, 5 mm; SN: USCN004202; Agilent). high-performance liquid chromatography (HPLC) (17,18) are typically used to detect ingredients in Chinese formu- Reagents lated products because of their outstanding separation Chlormycetin, glucuronidase, and aryl vitriol acetum power and automatable nature. However, owing to the enzyme were obtained from Sigma Chemical Co. (USA). All presence of multiple components in blood samples, EMs selected EMs and chlormycetin had chemical purity §98% cannot be readily quantified using GC and HPLC, which and were applied without further purification. Methanol, discriminate by retention time only. acetonitrile, toluene (chromatographic-grade purity), glucur- HPLC-electrospray ionization-tandem mass spectrome- onidase/aryl vitriol acetum enzyme solution, ammonium try (HPLC-ESI-MS/MS) offers a variety of advantages acetate, acetic acid, methyl tertiary butyl ether (MTBE), and over these methods for the quantification of serum EMs. acetidin were obtained from Merck & Co., Inc. (USA).

Figure 2. Chemical structure, molecular formula (MF), and molecular mass (MM) of the 6 estrogen metabolites.

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Preparation of standard solutions if a definite osteophyte was observed in the symptomatic Stock solutions of 6 EMs (Figure 2) and chlormycetin knee on plain radiographs, in accordance with American were each prepared at 80 mg/mL by dissolving 2 mg of College of Rheumatology criteria for OA (22). Exclusion each EM or chlormycetin powder in methanol to a final criteria for patients included: nonsteroidal anti-inflammatory volume of 25 mL in a volumetric flask. Stock solutions were drugs 3 days prior to the investigation; therapy monitored for time-dependent degradation by measuring during the 6 months prior to the investigation; use of con- the absolute peak height of each EM and chlormycetin. The traceptives, weight-reducing aids, , or any hor- stock solutions were stable for at least 50 days if stored at mone-replacement therapy; recent history of medication, –206C. Standard solutions were prepared by diluting malignancy, hypertension, diabetes, viral hepatitis, paren- 1.0 mL of each EM stock solution in methanol to a final chyma diseases, or fatty liver. Exclusion criteria for volume of 100 mL. healthy pre- or postmenopausal women included a history Calibration standards (1, 5, 10, 20, 40, and 80 ng/mL) of OA, smoking and alcohol use, and current . were prepared by diluting standard solutions in methanol. Internal standard solutions were prepared by diluting Blood sample collection 0.25 mL of chlormycetin stock standard solution in methanol Venous blood was collected from premenopausal and to a final volume of 20 mL. postmenopausal OA patients and healthy women. In premenopausal subjects, blood samples were collected Acetate buffer solution preparation during the follicular (days 5-7) and luteal (days 22-25) Acetate buffer solution at 0.04 M was prepared by phases of the same menstrual cycle, whereas only one dissolving 43.0 g ammonium acetate in 200 mL water, and blood sample was drawn from postmenopausal subjects. then adding 25.2 g glacial acetic acid. The pH was adjusted Menstrual and menopausal status was self-reported. to 5.0 and the final volume was brought to 1000 mL using Menopausal status was defined as premenopausal (women water. having regular menses) or postmenopausal (no menses in the last 12 months). Blood was allowed to clot for 1 h at Subjects room temperature, then centrifuged at 300 g for 10 min and All subjects were Asian. Baseline characteristics frozen at– 806C until assayed. included age, disease duration, body height, body weight, and body mass index (Table 1). Thirty-two premenopausal Sample preparation procedure outpatients (aged 37-47 years) and 32 postmenopausal Internal standard solutions (50 mL) were added to a 0.5- inpatients (aged 55-68 years) with OA of the knee were mLaliquotofserum,afterwhich100mL freshly prepared selected as the experimental groups, and 48 healthy glucuronidase/sulfatase and 2.0 mL 0.04 M acetate buffer women, including 24 premenopausal women (aged 37-46 solutions were added. After vortexing, the sample was years) and 24 postmenopausal women (aged 56-69 years), transferred to an incubator and hydrolyzed at 376Cfor4-8h. participated as the control group. The length of time since After hydrolysis, 5 mL acetidin was added to the mixture, menopause in all experimental and control postmenopausal which was then placed in the supersonic generator for women was in excess of 5 years. All patients were from the 5 min. After centrifuging at 12,000 g for 10 min, the super- Linyi People’s Hospital (Shandong, China). OA patients natant (organic liquid phase) was transferred to a rotary were from the Orthopedics Department, and healthy controls evaporation bottle, and the remaining solution was extracted were from the Medical Examination Center. All subjects with acetidin. The supernatant was then mixed and evapo- gave written informed consent and the study was approved rated to dryness in a rotary evaporation apparatus at 406C, by the Institute Ethics Committee of Linyi People’s Hospital. after which 0.5 mL methanol was added to dissolve the Patients reporting knee pain and stiffness (time extract. Samples were analyzed by HPLC-ESI-MS/MS ,30 min) or crepitus were diagnosed as symptomatic OA after 0.45-mm filtering (Millipore). Samples for free serum

Table 1. Baseline characteristics of subjects.

Follicular phase Luteal phase Postmenopausal phase

OA (n=32) Control (n=24) OA (n=32) Control (n=24) OA (n=32) Control (n=32)

Age (years) 41.8 ± 5.7 43.4 ± 7.4 41.8 ± 5.7 43.4 ± 7.4 60.6 ± 6.4 56.9 ± 3.0 Disease duration 4.6 ± 2.0 NA 4.6 ± 2.0 NA 16.3 ± 2.2 NA Body height (cm) 156.2 ± 0.7 151.2 ± 0.6 156.2 ± 0.7 151.2 ± 0.6 154.8 ± 0.6 159.1 ± 0.8 Body weight (kg) 59.2 ± 5.4 56.7 ± 6.4 59.2 ± 5.4 56.7 ± 6.4 71.5 ± 4.9 69.1 ± 6.5 BMI (kg/m2) 24.2 ± 1.7 23.0 ± 1.8 24.2 ± 1.7 23.0 ± 1.8 30.5 ± 1.4 27.6 ± 3.4

Data are reported as means±SD. OA: osteoarthritis; control: healthy women; BMI: body mass index; NA: not applicable.

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Table 2. Mass spectrometry parameters of HPLC-ESI-MS/MS EM measurement were processed as described earlier, with measurements of estrogen metabolites and chlormycetin. the exclusion of the glucuronidase/sulfatase hydrolysis step.

Q1/Q3 DP FP EP CE CXP Calibration standards preprocessing Internal standard solutions (50 mL) were added to each E 269/145 121 219 11 50 34 1 of the calibration standards (1, 5, 10, 20, 40, and 80 ng/mL), E 271/183 30 200 10 55 15 2 following the addition of 100 mL freshly prepared glucu- E 287/145 121 350 10 60 21 3 ronidase/sulfatase and 2.0 mL 0.04-M acetate buffer 2-OHE 285/160 121 219 11 52 15 1 solution. After vortexing, the samples were transferred to 2-OHE 287/147 121 219 11 60 15 2 an incubator at 376Cfor4h. 16a-OHE1 285/145 85 350 10 54 23 Chlormycetin 320/257 30 300 10 21 15 HPLC-ESI-MS/MS analysis Q1/Q3: parent ion/daughter ion; DP: declustering potential; FP: HPLC-ESI-MS/MS analysis was performed using an focusing potential; EP: entrance potential; CE: collision energy; API3000 tandem quadrupole axle mass spectrometer with CXP: collision cell exit potential. E1: estrone; E2: estradiol; E3: a Turbo spray ESI ionization source. Data were processed estriol; 2-OHE1: 2-hydroxyestrone; 2-OHE2: 2-hydroxyestradiol; using an Analyst 1.4 workstation. HPLC samples (10 mL) 16a-OHE1: 16a-hydroxyestrone. were injected on a ZORBAX C18 chromatographic column,

Figure 3. Product mass spectrum of EMs and internal standards. E1: estrone; E2: estradiol; E3: estriol; 2-OHE1: 2-hydroxyestrone; 2-OHE2:2- hydroxyestradiol; 16a-OHE1: 16a-hydroxyes- trone; chlormycetin.

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16a-OHE1, or chlormycetin. In negative ion mode, mass spectra were typical and specific for each reference substance (Figure 3).

Selection of color spectrum behavior and diagnosis ions The most sensitive and characteristic parent ion and daughter ion of each reference substance were selected to compose an ion pair, which were then measured after separating with methyl cyanide solution. We found that a Figure 4. Total ion flow chart. E1: estrone; E2: estradiol; E3: estriol; 2-OHE1: 2-hydroxyestrone; 2-OHE2: 2-hydroxyestradiol; fixed-proportion mobile phase was unsuitable for quanti- 16a-OHE1: 16a-hydroxyestrone. tative analysis because it failed to separate the 6 EM reference substances effectively owing to the width of and maintained at 356C. The mobile phase consisted the chromatographic peaks. We subsequently established of methyl cyanide solution and operated at a flow rate of chromatographic conditions in which each reference sub- 200 mL/min. For the analysis of EMs, a linear gradient was stance was completely separated with satisfactory signal established that changed the methyl cyanide/water solvent intensity (Figure 4). ratio from 20/7 to 20/13 over 15 min. The ionizing voltage was –4200 V in the negative ion mode, the ion pair of the Selection of extraction solvent parent ion (Q1)/daughter ion (Q3) was set up as the unit of We tested the extraction efficiency of alcohol, acetone, discernment, and the dwell time of each ion pair was ethylether, acetidin, MTBE, and chloroform with respect to 150 ms. Other parameters are listed in Table 2. the selected EMs and chlormycetin, and found that MTBE was the most efficient (data not shown). Statistical analysis Normal distributions were validated using the Methodology index analysis Kolmogorov-Smirnov method. Intergroup comparisons The precision, recovery, and other methodology indices were carried out using an independent-samples t-test to for each ion pair are listed in Table 3. The detection limit was determine whether experimental and control values in defined as the concentration corresponding to a level 10 OA and healthy women were significantly different. All times above the level of acoustic noise when a blank sample statistical analyses were performed using the SPSS was added to each progressively diluted reference sub- software (USA). Type I error was assessed based on stance solution. Precision was measured from the relative two-sided tests. P values less than 0.05 were considered standard deviation of the mean of 11 continuous measure- to be significant. ments after adding 1.0 ng internal standard to each sample. Accuracy was defined as the mean value of each sample of Results 20 replicates with known concentrations, in which 1.0 ng internal standard was added. Recovery was calculated Characteristic ion pair options of MS/MS using the formula: P (%)=(Call–Csample)/Caddition6100. For the phenolic hydroxyl group of estrogens as proton donors, the signal is known to be more sensitive in the Serum levels of selected estrogens and estrogen negative ion mode than in the positive ion mode. In this metabolites study, an exteriorized needle pump mode was used Compared to healthy women, serum-free E2 was for each standard solution of E1,E2,E3,2-OHE1, 2-OHE2, lower (P,0.05) in the luteal and postmenopausal phases

Table 3. Methodology index of estrogen metabolite analyses.

Linear range Linear equation r Precision (%) Recovery (%) Detection limit (ng/mL) (pg)

E1 0.2-10000 y = 0.01126 x – 0.01701 0.9994 2.6 98.8 10

E2 0.2-10000 y = 0.00778 x +0.00104 0.9998 1.3 103.4 10

E3 0.7-10000 y = 0.00434 x – 0.00229 0.9997 2.1 97.6 15

2-OHE1 1.0-10000 y = 0.00835 x +0.03071 0.9995 1.8 107.4 10

2-OHE2 0.5-10000 y = 0.00634 x +0.03022 0.9999 1.1 99.4 15

16a-OHE1 0.2-10000 y = 0.001132 x +0.02173 0.9991 3.1 105.4 10 r: correlation coefficient; E1: estrone; E2: estradiol; E3: estriol; 2-OHE1: 2-hydroxyestrone; 2-OHE2: 2-hydroxyestradiol; 16a-OHE1: 16a- hydroxyestrone.

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Table 4. Serum free (unconjugated) and total (conjugated + unconjugated) levels of selected estrogen metabolites in the follicular, luteal, and postmenopausal phases in women with OA and healthy controls.

Follicular phase Luteal phase Postmenopausal phase

OA (n=32) Control (n=24) OA (n=32) Control (n=24) OA (n=32) Control (n=24)

Free

E1 52.5 ± 21.7 54.1 ± 11.9 48.0 ± 6.6 51.3 ± 2.7 28.2 ± 9.4 31.9 ± 4.8

E2 71.5 ± 14.2 75.1 ± 19.1 65.0 ± 9.2* 72.3 ± 3.2 13.7 ± 3.1* 18.2 ± 5.6

E3 15.4 ± 4.8 14.7 ± 1.8 16.6 ± 2.0 17.4 ± 1.4 5.9 ± 1.4 7.0 ± 1.1 Total

E1 643.0 ± 310.5 670.9 ± 113.4 726.6 ± 42.0 756.5 ± 68.8 404.1 ± 62.5 426.6 ± 34.7

E2 117.9 ± 14.5 123.8 ± 28.8 141.5 ± 9.3 149.4 ± 18.5 40.1 ± 8.0** 51.4 ± 12.1

E3 48.0 ± 18.2 51.8 ± 11.6 49.5 ± 7.0 47.0 ± 6.2 24.7 ± 8.0 27.0 ± 5.9

2-OHE1 262.7 ± 45.4* 304.1 ± 51.5 264.2 ± 40.4* 296.5 ± 31.1 64.1 ± 9.2 67.3 ± 8.4

2-OHE2 27.1 ± 7.9 24.0 ± 7.8 26.3 ± 8.4 23.9 ± 7.2 16.9 ± 4.9** 11.6 ± 4.0

16a-OHE1 23.1 ± 5.3 25.4 ± 6.7 22.1 ± 2.3 23.4 ± 1.6 11.6 ± 2.0 13.1 ± 1.2

Data are reported as means±SD in pg/mL. OA: osteoarthritis; control: healthy women; E1: estrone; E2: estradiol; E3: estriol; 2-OHE1: 2-hydroxyestrone; 2-OHE2: 2-hydroxyestradiol; 16a-OHE1: 16a-hydroxyestrone. * P,0.05, OA compared to control; **P,0.01, OA compared to control (t-test).

in women with OA, and total serum E2 was lower 2-OHE1, and in raising levels of 2-OHE2. (P,0.01) in postmenopausal women with OA (Table 4). Our HPLC-ESI-MS/MS assay had a detection limit of 10- Furthermore, compared to healthy women, total serum 2- 15 pg/mL serum and a precision of 1.1-3.1%, enabling us to OHE2 was higher (P,0.01) in postmenopausal women measure endogenous serum estrogens in pre- and post- with OA and total serum 2-OHE1 was lower (P,0.05) in menopausal women. The identification and quantification of both the luteal and follicular phases in women with OA. serum estradiol levels in the low pre- and postmenopaus- Neither free nor total serum levels of E1 and E3 in pre- and al range (,30 pg/mL) are important prognostic tools for postmenopausal women with OA differed significantly from common chronic diseases of women (27). RIA methods are those in healthy subjects. insufficiently accurate or sensitive to monitor serum estradiol at such low levels (28-30), and, although bioassays using Discussion recombinant yeast and HeLa cells are more sensitive than RIA, they lack specificity and convenience (31). Moreover, OA involves the progressive degeneration of articular whereas GC/tandem mass spectrometry is sufficiently cartilage and structural changes in the underlying bone, specific, sensitive, and accurate, it lacks the precision of our including the development of marginal growths and osteo- HPLC-ESI-MS/MS method (32-34). The MS-based detection phytes, and thickening of the periosteum (23). A century ago, of derivatized estrogens in multiple samples using the cation the description by Cecil and Archer (24) of ‘‘arthritis of the mode of analysis has been recently reported (35). Although menopause,’’ referring to the development of hand and knee this approach reportedly raised the sensitivity of analysis, the OA during menopause, suggested that estrogen deficiency efficiency of derivation was not investigated in a previous might play a role in OA. Subsequent epidemiological studies study (36), suggesting that the derivation procedure might have indicated that the incidence and prevalence of hip, introduce errors. In addition, the isotope-labeled estrogens knee, and finger OA are greater in men than in women and EM standards used in previous studies (22,37,38) are before 50 years of age, after which age they are increasingly more expensive than our negative ion mode MS/MS assay. more common in women compared to men (25). The free serum E2 levels in pre- and postmenopausal The expression of estrogen receptors in articular carti- women detected in the current study (Table 4) were lage (26) suggests that, in addition to estradiol deficiency, statistically similar to those obtained previously (23), with 2-OHE1 and 2-OHE2 might be involved in the etiology of four-fold lower standard deviations and a six-fold greater female OA and that manipulation of their levels has potential number of samples from postmenopausal women. in the prevention and cure of this condition. In this regard, In conclusion, we have established an HPLC-ESI-MS/ future studies are required to determine the specific cell MS method for the simultaneous detection of serum levels types, such as chondrocytes, synoviocytes, and bone cells, of 6 EMs in pre- and postmenopausal women with OA that are involved in reducing levels of 17b-estradiol (E2)and and healthy controls. Because the MS/MS was performed

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in multiple reaction-monitoring tandem quadrupole mass Acknowledgments spectrometry using ESI in negative ion mode, blood samples derivatives were not necessary, making the assay Research supported by the Excellent Young Scientist more economical, rapid, and efficient. Accordingly, our Research Award fund of Shandong China (#BS2011YY040), HPLC-ESI-MS/MS method has potential clinical applica- the Medical and Health Science and Technology De- tions, as well as for treatment or intervention studies velopment Plan of Shandong China (#2011QW009), requiring precise measurements of fluctuations in EM levels, and the development projects of Shandong Province such as in perimenopausal women, or measurement of low Science and Technology of Traditional Chinese Medicine analyte concentrations. In this regard, further improvements (#2013ZDZK155). are required to minimize variability in measurements.

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